Project Summary/Abstract: Carbapenem-Resistant Enterobacteriaceae (CRE) have been classified as an urgent public health threat in the US and around the globe. New Delhi Metallo-?-lactamases (NDM) producing CRE are particularly concerning as they have rapidly spread worldwide and can efficiently co- exist with a plethora of Gram-negative resistance determinants including Extended Spectrum ?-lactamases (ESBLs), carbapenemases, and polymyxin resistance genes. We have reported the first US case of polymyxin- and carbapenem-resistant E. coli producing New Delhi Metallo-beta-lactamase (NDM-5) together with mobile colistin resistance (MCR-1) in a patient. The recent report of pan-drug-resistant (PDR), K. pneumoniae (NDM-1, ESBLs, and polymyxin resistance determinants), from a patient in Nevada further highlights that it may be only a matter of time until hospitals in the US and worldwide face an outbreak of these Gram-negative ?superbugs?. It is critical to prepare therapeutics for the future occurrence of NDM strains which harbor a diverse array of resistance determinants. Our Central Hypothesis is that rationally optimized antibiotic combination dosing strategies will achieve extensive killing and prevent emergence of resistance against of NDM-producing Enterobactericeae. Our preliminary studies provide compelling evidence in support of our innovative combinations. We established the first highly efficient cassette assay to assess target site penetration of ?-lactams in the presence of polymyxins, the first dataset on ?-lactam receptor binding in K. pneumoniae, and show that new 4-drug combination regimens eradicated NDM and ESBL co-producing K. pneumoniae and prevented resistance. In Aim 1, we will create genetically engineered strains, as well as assess the target site penetration and receptor binding of ?-lactam antibiotics and ?-lactamase inhibitors, and the enhanced penetration in presence of polymyxins. In Aim 2, in vitro pharmacokinetic/pharmacodynamics models, including the dynamic Hollow Fiber Infection Model, will evaluate optimized dosing strategies for 3- and 4-drug combinations by profiling the time course of bacterial killing, suppression of resistance, and persister eradication. Genomics and transcriptomics will be utilized to understand why monotherapies and non-optimized combinations failed with resistance. In Aim 3, our latest Quantitative and Systems Pharmacology (QSP) modelling approach will guide translation across all experimental tiers. Prospective validation of these novel optimal combination dosing strategies will be completed in murine pneumonia models with an intact and impaired immune system. This will yield innovative combination dosage regimens against pandrug-resistant CRE that can suppress resistance. Thus, this project will address an urgent, global medical need. This project will provide the first mechanistically informed, rationally optimized and prospectively validated combination dosing strategies of available antibiotics against resistant Gram-negatives that will be ready for testing in future clinical trials.